An Intracellular Arrangement of Histoplasma capsulatum Yeast-Aggregates Generates Nuclear Damage to the Cultured Murine Alveolar Macrophages

Histoplasma capsulatum is responsible for a human systemic mycosis that primarily affects lung tissue. Macrophages are the major effector cells in humans that respond to the fungus, and the development of respiratory disease depends on the ability of Histoplasma yeast cells to survive and replicate within alveolar macrophages. Therefore, the interaction between macrophages and H. capsulatum is a decisive step in the yeast dissemination into host tissues. Although the role played by components of cell-mediated immunity in the host's defense system and the mechanisms used by the pathogen to evade the host immune response are well understood, knowledge regarding the effects induced by H. capsulatum in host cells at the nuclear level is limited. According to the present findings, H. capsulatum yeast cells display a unique architectural arrangement during the intracellular infection of cultured murine alveolar macrophages, characterized as a formation of aggregates that seem to surround the host cell nucleus, resembling a crown. This extranuclear organization of yeast-aggregates generates damage on the nucleus of the host cell, producing DNA fragmentation and inducing apoptosis, even though the yeast cells are not located inside the nucleus and do not trigger changes in nuclear proteins. The current study highlights a singular intracellular arrangement of H. capsulatum yeast near to the nucleus of infected murine alveolar macrophages that may contribute to the yeast’s persistence under intracellular conditions, since this fungal pathogen may display different strategies to prevent elimination by the host's phagocytic mechanisms

Antifungal Activity of Decyl Gallate against Several Species of Pathogenic Fungi

This work aims to demonstrate that the gallic acid structure modification to the decyl gallate (G14) compound contributed to increase the antifungal activity against several species of pathogenic fungi, mainly, Candida spp., Cryptococcus spp., Paracoccidioides spp., and Histoplasma capsulatum, according to standardized microdilution method described by Clinical Laboratory Standard Institute (CLSI) documents. Moreover this compound has a particularly good selectivity index value, which makes it an excellent candidate for broad-spectrum antifungal prototype and encourages the continuation of subsequent studies for the discovery of its mechanism of action

Análise proteômica diferencial do biofilme de histoplasma capsulatum e implicações na interação fungo-hospedeiro

Histoplasma capsulatum var. capsulatum é um fungo dimórfico que causa a histoplasmose, uma micose respiratória e sistêmica. H. capsulatum é primariamente adquirido após a exposição ao aerosol, pela inalação de microconídeos ou fragmentos de hifas. A evolução da doença respiratória depende da capacidade da levedura de Histoplasma em sobreviver e se replicar dentro dos macrófagos alveolares. Neste contexto, é conhecido que a adesão às células do hospedeiro constitui o primeiro passo para a colonização e é essencial para o estabelecimento da infecção. Em vista disso, alguns microrganismos aderem às superfícies biológicas e não biológicas formando biofilmes. Biofilmes são comunidades dinâmicas de microrganismos, que aderidos às superfícies, produzem uma matriz exopolimérica e essa formação representa um estado que permite que as células microbianas sobrevivam em ambientes hostis e dispersem para colonizar novos nichos. Com base na importância potencial de biofilmes para sua sobrevivência no hospedeiro humano e na natureza, este trabalho foi desenvolvido para investigar pela primeira vez a formação de biofilme por H. capsulatum correlacionando com sua capacidade em aderir às células epiteliais, bem como definir particularidades da interação entre macrófagos e H. capsulatum, incluindo análise de fragmentação nuclear pelo ensaio do cometa e TUNEL. Os ensaios foram realizados in vitro com duas cepas clínicas de H. capsulatum, uma isolada no México e a outra no Brasil. Além disso, foi realizada análise proteômica diferencial do fungo em biofilme e sob a condição planctônica. Os resultados mostraram que H. capsulatum é capaz de formar biofilmes e ambos os isolados selecionados tem uma alta capacidade em aderir aos pneumócitos (A549). Através de análise proteômica, o isolado EH-315 exibiu diferentes perfis protéicos, com aproximadamente...Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a respiratory and systemic mycosis. H. capsulatum is primarily acquired after exposure to the aerosol, by microconideas or hyphal fragments inhalation. The development of a respiratory disease depends on the ability of Histoplasma yeast to survive and replicate within alveolar macrophages. In this context, it is known that the adhesion to host cells is the first step in the colonization and is essential for the establishment of the infection. As a result, some microorganisms adhere to biological and non-biological surfaces producing biofilms. Biofilms are dynamic communities of microorganisms that adhere to surfaces producing an exopolimeric matrix, and this formation is a state that allows microbial cells to survive in hostile environments and to disperse to colonize new niches. Based on the potential importance of biofilms to survive in the human host and in nature, this study was performed to investigate, for the first time, the biofilm formation by H. capsulatum correlating with their ability to adhere to epithelial cells, as well as defining features of the interaction between macrophages and H. capsulatum, including analysis of nuclear fragmentation by comet assay. In addition, we performed differential proteomic analysis of the fungus in biofilm and under planktonic conditions. The results demonstrated that H. capsulatum is able to form biofilms and both selected isolates have a high ability to adhere to pneumocytes (A549). Through proteomic analysis, the EH-315 isolate exhibited different protein profiles, with approximately 250 proteins exclusively expressed in the biofilm format and others with different levels of expression when compared to the fungus in planktonic growth. The sequencing by MALDI-TOF/TOF revealed that most proteins are nuclear or are involved in the metabolism... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Role of paracoccin on Paracoccidioides brasiliensis virulence and susceptibility to antifungal drugs in the Galleria mellonella larvae model

ABSTRACTParacoccin (PCN), a Paracoccidioides brasiliensis glycoprotein, has been reported to play roles in fungal biology and paracoccidioidomycosis pathogenesis. Lectin and chitinase domains account for the PCN’s dual roles as an immunomodulatory agent and virulence factor. Soluble PCN injected in P. brasiliensis infected mice, by interacting with TLRs’ N-glycans, drives the host immune response toward a protective Th1 axis. Otherwise, mice infection with yeasts overexpressing PCN (ov-PCN) revealed that PCN acts as a fungal virulence factor, thanks to its chitinase activity on the cell wall, resulting in resistance to phagocytes’ fungicidal activity and development of severe paracoccidioidomycosis. Because antifungal drug administration follows the disease diagnosis, we studied the PCN effect on yeast resistance or susceptibility to antifungal agents. Using a paracoccidioidomycosis model developed in Galleria mellonella larvae, we confirmed the observation, in the murine host, that ov-PCN yeasts display maximum virulence compared to wild-type (wt-PCN) or PCN-silenced (kd-PCN) yeasts. PCN overexpression accounted for the highest susceptibility of P. brasiliensis to antifungal and reduced relative mRNA expression of genes encoding proteins related to cell wall remodeling. The lowest virulence, detected in infection with kd-PCN yeasts, correlated with the lowest susceptibility to antifungals and impact on genes for cell wall remodeling. So, we defined that the grade of endogenous PCN production influences the P. brasiliensis virulence and susceptibility to antifungal drugs, as well as the expression of genes related to cell wall remodeling. We postulate that this variable gene expression is mechanistically associated with P. brasiliensis virulence changes

A mini review of Candida species in hospital infection: epidemiology, virulence factor and drugs resistance and prophylaxis

The introduction of more efficient diagnostic methods, new techniques in surgery and transplantation, antibiotics and chemotherapeutics more potent and novel materials for prostheses, catheters and probes significantly increased the life expectancy and quality of life of critically ill patients, on the other hand, hospital-acquired infections emerged as important iatrogenic complications. Invasive infections are a growing problem in public health hospitals in Brazil and worldwide. Among the various etiological agents found in the hospital environment, the genus Candida has been the third most frequently isolated pathogen. In general, invasive fungal infections are associated with high morbidity and mortality, difficulties in diagnosis, antimicrobial resistance, length of hospital stay and increased hospital costs. This mini review of the literature describes about epidemiology of hospital infection of Candida species, as well as its virulence factors and drugs resistanc

Highlights in pathogenic fungal biofilms

A wide variety of fungi have demonstrated the ability to colonize surfaces and form biofilms. Most studies on fungal biofilms have focused on Candida albicans and more recently, several authors have reported the involvement of other genera of yeasts and Candida species, as well as of filamentous fungi in the formation of biofilms, including: Cryptococcus neoformans, Cryptococcus gattii, Rhodotorula species, Aspergillus fumigatus, Malassezia pachydermatis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Pneumocystis species, Coccidioides immitis, Fusarium species, Saccharomyces cerevisiae, Trichosporon asahii, Mucorales and Blastoschizomyces. There is a current interest in describing the particular characteristics of the biofilm formation by of these fungi. A major concern is the control of biofilms, requiring knowledge of the biofilm mechanisms. However, our knowledge of these microbial communities is limited, due to the complexity of these systems and metabolic interactions that remain unknown. This mini-review aims to highlight recently discovered fungal biofilms and to compare them with the current knowledge on biofilms.This manuscript is part of the series of works presented at the V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi (Oaxaca, Mexico, 2012). (C) 2013 Revista Iberoamericana de Micologia. Published by Elsevier Espana, S.L. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Genetic evaluation and activity of antifungal against clinical isolate Candida albicans biofilms

Candida yeasts are common in the oral cavity and can cause candidosis in the presence of predisposing factors, especially diabetes. The manifestation of the disease is related to this set of local factors such as the presence of dental prostheses, salivary pH, salivary flow and tobacco and the ability to form biofilms. Biofilms are specific and organized communities of cells under the control of signaling molecules rather than random accumulations of cells resulting from cell division and frequently are drugs resistance. Aim: The objectives of this study were to determine the genetic patterns of these C. albicans isolates and to evaluate the in vitro activity amphotericin B and caspofungin against C. albicans biofilms. Methods: Microbial samples were collected from subgingival sites and seeded in CHROMagar for subsequent identification of C. albicans by PCR. Genotypes were defined based on the identification of the transposable introns in the 25S rDNA by PCR. Results: In this study, 6 strains were identified as C. albicans and of these, 3 strains were genotype A and 3 were genotype B. The results showed that both amphotericin B and caspofungin exhibited strong antifungal activities against C. albicans biofilm formation and inhibiting the biofilm formation ranging from 70.8 – 95.3% and 77.7 - 88.7%, respectively. The antifungals studied had low inhibitory effect on preformed biofims, ranging from 39.5 - 50.8% for amphotericin B and from 23.1 - 36.9% for caspofungin at the same concentration. The activity of the two drugs was most effective in inhibit biofilm formation